Allergen peptide fragments and use thereof for treatment of dust mite allergies

ABSTRACT

The present invention relates generally to in vivo methods and compositions designed for allergen specific immunotherapy. The compositions include contiguous overlapping peptide fragments which together form an entire amino acid sequence of an allergen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 13/284,383 filed Oct.28, 2011 which is a divisional of U.S. application Ser. No. 11/226,162filed Sep. 14, 2005, now U.S. Pat. No. 8,075,897, which is acontinuation-in-part of International Application No. PCT/IB2004/01300filed Mar. 15, 2004, which is a continuation of U.S. application Ser.No. 10/799,514 filed Mar. 12, 2004, now U.S. Pat. No. 7,923,209, whichin turn claims the priority benefit under 35 U.S.C. §119(e) of U.S.provisional application Ser. No. 60/455,004 filed Mar. 14, 2003 thedisclosures of which are hereby incorporated by reference. This also isa continuation-in-part of U.S. application Ser. No. 10/799,514 filedMar. 12, 2004, now U.S. Pat. No. 7,923,209, which in turn claims thepriority benefit under 35 U.S.C. §119(e) of U.S. provisional applicationSer. No. 60/455,004 filed Mar. 14, 2003 the disclosures of which arehereby incorporated by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablesequence listing submitted concurrently herewith and identified asfollows: File Name: 41486E_SeqListing.txt; Size: 25,900 bytes; CreatedMar. 11, 2014

FIELD OF THE INVENTION

The present invention relates generally to in vivo methods andcompositions designed for allergen-specific immunotherapy. Thecompositions include contiguous overlapping peptide fragments whichtogether comprise the entire amino acid sequence of an allergen.

BACKGROUND OF THE INVENTION

IgE-mediated allergies represent a major health problem in theindustrialized world. The immediate symptoms of the disease (e.g.allergic rhinoconjunctivitis, dermatitis, bronchial asthma, anaphylacticshock) are caused by the cross-linking of effector cell-bound IgEantibodies by allergens, which leads to the release of biologicalmediators such as histamine or leukotrienes. In order to induce strongeffector cell activation, and thus inflammatory responses, an allergenmust be able to cross-link effector cell-bound IgE antibodiesefficiently.

Allergy immunotherapy (allergy shots) is a treatment that involvesinjections of small amounts of the allergens to which a person isallergic. Over time, the concentration of the injections is increased,which leads to the production of blocking antibodies (called IgGantibodies, mainly IgG4 antibodies in humans) and a decrease in thelevel of allergic antibodies (IgE antibodies). In this way immunity isdeveloped (e.g., a person may require allergy immunotherapy againstgrass, weed and tree pollens, house dust mites, cat and dog dander andinsect stings).

This form of treatment varies in efficacy among different types ofallergy and between individuals. Pollen, dust mite, dander and insectvenom allergic reactions usually respond well. Current research involvesdetermining exactly which mechanisms are active in a specific patient soallergen immunotherapy is better tailored to the individual. Also, workis ongoing to better define chemically the allergens used for treatment,to make allergen immunotherapy safer, and to safely increase theinterval between injections.

Immunologic mechanisms of desensitization are still incompletelyunderstood, although they appear to be associated with a Th2 to Th1cytokine shift, with a decrease in the levels of allergen-specific IgE,and with a marked decrease in T cell response to the allergen,eventually leading to T cell tolerance (Secrist et al., J. Exp. Med.178:2123, 1993; Jutel et al., J. Immunol. 154:4187, 1995; Kammerer etal., J. Allergy Clin. Immunol. 100:96, 1997; Akdis et al., FASEB J.13:603, 1999; and Muller et al., J. Allergy Clin. Immunol 101:747,1998). This may, directly or indirectly, contribute to decreased mastcell or eosinophil activation and may also improve patient protectionupon reexposure to the allergen (Jutel et al., Clin. Exp. Allergy26:1112 1996).

Safer methods of immunotherapy with reduced risk of anaphylaxis need tobe developed.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 1, 2and 3) which together comprise the entire amino acid sequence of a beevenom allergen (SEQ ID NO: 4), wherein the fragments are capable ofinducing a T cell response in patients who are hypersensitive to theallergen.

In another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 5 and6) which together comprise the entire amino acid sequence of a birchpollen allergen (SEQ ID NO: 7), wherein the fragments are capable ofinducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 8 and9) which together comprise the entire amino acid sequence of a birchpollen profilin allergen (SEQ ID NO: 10), wherein the fragments arecapable of inducing a T cell response in patients who are hypersensitiveto the allergen.

In a further aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 11,12 and 13) which together comprise the entire amino acid sequence of adust mite allergen (SEQ ID NO: 14), wherein the fragments are capable ofinducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 15and 16) which together comprise the entire amino acid sequence of a dustmite allergen (SEQ ID NO: 17), wherein the fragments are capable ofinducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 5 and8) which together comprise the entire amino acid sequence of a chimericbirch pollen allergen (SEQ ID NO: 18), wherein the fragments are capableof inducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 9 and6) which together comprise the entire amino acid sequence of a chimericbirch pollen allergen (SEQ ID NO: 19), wherein the fragments are capableof inducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 8 and5) which together comprise the entire amino acid sequence of a chimericbirch pollen allergen (SEQ ID NO: 20), wherein the fragments are capableof inducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 6 and9) which together comprise the entire amino acid sequence of a chimericbirch pollen allergen (SEQ ID NO: 21), wherein the fragments are capableof inducing a T cell response in patients who are hypersensitive to theallergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 15and 11) which together comprise the entire amino acid sequence of achimeric dust mite allergen (SEQ ID NO: 22), wherein the fragments arecapable of inducing a T cell response in patients who are hypersensitiveto the allergen.

In yet another aspect, the invention provides a composition containing aplurality of contiguous overlapping peptide fragments (SEQ ID NOs: 13and 16) which together comprise the entire amino acid sequence of achimeric dust mite allergen (SEQ ID NO: 23), wherein the fragments arecapable of inducing a T cell response in patients who are hypersensitiveto the allergen.

Preferably, administration of the compositions of the invention resultsin lower levels of IgE stimulation activity. More preferably,administration results in weak or zero IgE stimulation activity (e.g.weak IgE binding or no IgE binding). As used herein, weak IgE bindingrefers to IgE production and/or cross-linking that is less than theamount of IgE production and/or IL-4 production stimulated by the wholeprotein allergen. Preferably, the compositions of the invention do notinduce immediate skin reactivity (wheal <5 mm with no flare) wheninjected intradermally at a concentration ≦1 μg/ml. Most preferably,administration of the compositions of the invention results in adecrease in T cell response upon subsequent exposure to the proteinallergen, thereby modulating an immune response of a patient against theprotein allergen.

In another aspect, the invention provides in vivo methods of determiningthe dose of composition needed to desensitize a patient to a specificallergen by introducing a series of compositions containing varyingconcentrations of a plurality of contiguous overlapping peptidefragments, which together comprise the entire amino acid sequence of theallergen, into the skin of the patient, wherein the fragments arecapable of inducing a T cell response in patients who are hypersensitiveto the allergen, further wherein said overlapping peptide fragments donot bind or weakly bind IgE; introducing a positive-control and anegative-control into the skin of the patient; checking for developmentof a wheal or flare at the introduction site; and comparing the size ofthe papule (<5 mm) and flare produced by the varying concentrations of aplurality of contiguous overlapping peptide fragments to thepositive-control and negative-control, thereby determining the dose ofcomposition needed to desensitize the patient to the specific allergen.For example, the patient is selected from the group consisting ofhumans, dogs, cats, pigs, horses, rats and mice. In one preferredembodiment, the patient is a human. In some embodiments, each peptide ofthe plurality of contiguous overlapping peptide fragments can be 30-90amino acids in length, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75,80, 81, 85, 86 and 90 amino acids in length. In various embodiments, theamino acid sequences of contiguous overlapping peptide fragments in theplurality overlap by from 5 to 25 amino acids with overlaps of 9 to 22amino acids being preferred with overlaps of from about 10 to about 15amino acids, e.g., 10, 11, 12, 13, 14 and 15 amino acids beingparticularly preferred.

The methods of the invention are useful in treating a number ofdifferent allergies to various allergens. For example, the allergensinclude, but are not limited to, plant pollens, grass pollens, treepollens, weed pollens, insect venom, dust mite proteins, animal dander,saliva, fungal spores and food allergens (i.e., peanut, milk, gluten andegg). In one embodiment, the allergen is insect venom. In one preferredembodiment, the insect venom is bee venom. The plurality of contiguousoverlapping peptide fragments may include SEQ ID NOs: 1, 2, and 3, whichcomprise the entire amino acid sequence of the major bee venom allergen(SEQ ID NO: 4). In another embodiment, the allergen is tree pollen. Inone preferred embodiment, the tree pollen is birch pollen. The pluralityof contiguous overlapping peptide fragments may include SEQ ID NOs: 5and 6, which comprise the entire amino acid sequence of the major birchpollen allergen (SEQ ID NO: 7). The plurality of contiguous overlappingpeptide fragments may include SEQ ID NOs: 8 and 9, which comprise theentire amino acid sequence of birch pollen profilin allergen (SEQ ID NO:10). In another embodiment, the allergen is dust mite proteins. Theplurality of contiguous overlapping peptide fragments may include SEQ IDNOs: 11, 12, and 13, which comprise the entire amino acid sequence ofthe dust mite allergen D. pteronyssinus 1 (SEQ ID NO:14). The pluralityof contiguous overlapping peptide fragments may include SEQ ID NOs: 15and 16, which comprise the entire amino acid sequence of the dust miteallergen D. pteronyssinus 2 (SEQ ID NO:17). The plurality of contiguousoverlapping peptide fragments may include at least two contiguousoverlapping peptide fragments selected from the group consisting of SEQID NOs: 1, 2, 3, 5, 6, 8, 9, 11, 12, 13, 15 and 16.

In various embodiments, the introducing is done by skin prick,intradermal or subcutaneous injection. Those skilled in the art willrecognize that any means of introducing can be employed. In someembodiments, the varying concentrations of contiguous overlappingpeptide fragments is from a concentration of about 0.001 μg/ml to about100 μg/ml. In preferred embodiments, the concentration of contiguousoverlapping peptide fragments are between 0.001-10.0, 0.01-10.0, or0.1-1.0 μg/ml.

In yet another aspect, the invention provides in vivo methods ofinducing tolerance in a patient allergic to a specific allergen byintroducing a plurality of contiguous overlapping peptide fragmentswhich together form an entire amino acid sequence of the allergen intothe skin of the patient, wherein the fragments are capable of inducing aT cell response in patients who are hypersensitive to the allergen,further wherein said overlapping peptide fragments do not bind or weaklybind IgE; and creating antibodies to the allergen, thereby buildingimmunity to the allergen, wherein the immunity leads to tolerance of theallergen in the patient. For example, the patient is selected from thegroup consisting of humans, dogs, cats, pigs, horses, rats and mice. Inone preferred embodiment, the patient is a human. In another embodiment,the antibodies created to the allergen are IgG antibodies. Morepreferably, the IgG antibodies are IgG4 antibodies. In some embodiments,each peptide of the plurality of contiguous overlapping peptidefragments can be 30-90 amino acids in length, e.g., 30, 35, 40, 45, 50,55, 60, 65, 70, 73, 75, 80, 81, 85, 86 and 90 amino acids in length. Invarious embodiments, the amino acid sequences of contiguous overlappingpeptide fragments in the plurality overlap by about 10 to about 15 aminoacids, e.g., 10, 11, 12, 13, 14 and 15 amino acids. In some embodiments,the varying concentrations of contiguous overlapping peptide fragmentsis from a concentration of about 0.001 μg/ml to about 1000 μg/ml. Inpreferred embodiments, the concentration of contiguous overlappingpeptide fragments are between 0.001-100.0, 0.01-10.0, or 0.1-1.0 μg/ml.

The methods of the invention are useful in treating a number ofdifferent allergies to various allergens. For example, the allergensinclude, but are not limited to, plant pollens, grass pollens, treepollens, weed pollens, insect venom, dust mite proteins, animal dander,saliva, fungal spores and food allergens (i.e., peanut, milk, gluten andegg). In one embodiment, the allergen is insect venom. In one preferredembodiment, the insect venom is bee venom. The plurality of contiguousoverlapping peptide fragments may include SEQ ID NOs: 1, 2, and 3, whichcomprise the entire amino acid sequence of the major bee venom allergen(SEQ ID NO: 4). In another embodiment, the allergen is tree pollen. Inone preferred embodiment, the tree pollen is birch pollen. The pluralityof contiguous overlapping peptide fragments may include SEQ ID NOs: 5and 6, which comprise the entire amino acid sequence of the major birchpollen allergen (SEQ ID NO: 7). In another preferred embodiment, theplurality of contiguous overlapping peptide fragments may include SEQ IDNOs: 8 and 9, which comprise the entire amino acid sequence of birchpollen profilin allergen (SEQ ID NO: 10). In one embodiment, theallergen is dust mite proteins. The plurality of contiguous overlappingpeptide fragments may include SEQ ID NOs: 11, 12, and 13, which comprisethe entire amino acid sequence of the dust mite allergen D.pteronyssinus 1 (SEQ ID NO:14). The plurality of contiguous overlappingpeptide fragments may include SEQ ID NOs: 15 and 16, which comprise theentire amino acid sequence of the dust mite allergen D. pteronyssinus 2(SEQ ID NO:17). The plurality of contiguous overlapping peptidefragments may include at least two contiguous overlapping peptidefragments selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5,6, 8, 9, 11, 12, 13, 15 and 16.

Preferably, the methods of the invention do not induce immediate skinreactivity (wheal <5 mm with no flare) when injected intradermally at aconcentration ≦1 μg/ml.

In various embodiments, the introducing is done by parenteral, e.g.,skin prick, intravenous, intradermal, subcutaneous, oral, nasal, mucosal(e.g., inhalation), transdermal (topical), transmucosal, lymph node andrectal administration. Those skilled in the art will recognize that anymeans of introducing can be employed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing PLA₂ derived overlapping peptide therapyinduces specific T cell anergy in bee venom hypersensitive patients.Hatched columns: peptide-treated group, open columns: control group(treated with albumin). Results are presented as plot-boxes and whiskerswith successive percentiles 5, 25, 50, 75, 95. Medians are indicated bythick bars.

FIG. 2 is a series of graphs showing overlapping peptide therapydeviates T cell cytokine response and strongly stimulates IL-10secretion. Cytokines (panel A: IL-4; panel B: IFN-γ; panel C: IL-10)from supernatants of short term T cell lines were measured by ELISA incell supernatant. Results are presented as plot-boxes and whiskers withsuccessive percentiles 5, 25, 50, 75, 95. Medians are indicated by thickbars.

FIG. 3 is a series of graphs showing anti-PLA₂ specific serum IgE.Anti-PLA₂ specific serum IgE were measured in peptide-treated group(panel A) and in control group (panel B) at the indicated time-points.Median values are indicated by thick bars.

FIG. 4 is a series of graphs showing anti-PLA2.specific serum IgG4.Anti-PLA2 specific serum IgG4 were measured in peptide-treated group(panel A) and in control group (panel B) at the indicated time-points.Median values are indicated by thick bars.

FIG. 5 is a photograph showing absence of immediate allergic reaction tooverlapping peptide fragments. A representative patient from the peptidegroup was injected intradermally with, respectively from left to right,0.01 μg/ml native PLA₂, 1 μg/ml of each of the three synthetic peptidefragments OPF₁₋₆₀, OPF₄₇₋₉₉ and OPF₉₀₋₁₃₄ separately and with a mixtureof them (1 μg/ml each) (arrows).

FIG. 6 is a series of graphs showing intradermal skin tests withpeptides and native PLA₂. Results are expressed as end-pointconcentrations (log₁₀ scale) at enrollment into the study (day 0) andafter the last injection at day 70 in patients from peptide group(panels A, B) and control group (panels C, D), tested respectively withthe three overlapping peptide fragments (as a mixture) (panels A, C) andwith native PLA₂ (panels B, D).

FIG. 7 is a series of graphs showing in vitro IgE binding to whole BVand native PLA₂ (panels A, B) and to overlapping peptide fragmentsOPF₁₋₆₀, OPF₄₇₋₉₉ and OPF₉₀₋₁₃₄ (panels C, D, E respectively), analyzedby dot blot assays after each injection. Results are expressed asabsorbance arbitrary units. Open columns: control group; hatchedcolumns: peptide group.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery that a plurality ofcontiguous overlapping peptide fragments can be used for allergenspecific immunotherapy. The use of a plurality of contiguous overlappingpeptide fragments for allergen immunotherapy induces both humoral andcellular responses comparable to native allergen rush immunotherapy.

Advantages of using the plurality of contiguous overlapping peptidefragments of the invention include, but are not limited to, theirability to induce a T helper cell response in hypersensitive patientsdue to the fact that they contain all possible T cell epitopes; theirability to efficiently recruit specific T cells, leading to a modulationof the immune response to allergens; and their ability to display lowIgE binding activity (they are hypoallergenic). Thus, the plurality ofcontiguous overlapping peptide fragments of the invention displaysignificantly reduced IgE binding activity, but conserved T cellactivating capacity, therefore making them ideal candidates for a noveland safe approach of specific immunotherapy.

Without being limited to any particular mechanism, the ability of aplurality of contiguous overlapping peptide fragments to induce a Thelper cell response in hypersensitive patients may be due to the factthat the amino acid sequence of contiguous overlapping peptide fragmentsin the plurality overlap by from about 5 to 25 amino acids and inparticular from 9 to 22 amino acids and from about 10 to about 15 aminoacids, e.g., 10, 11, 12, 13, 14 and 15 amino acids and cover multiple Tcell epitopes. Therefore these combinations of peptides do not require Tcell epitope customization to fit with each patient's majorhistocompatibility complex (MHC) molecules (HLA restriction). Theability of contiguous overlapping peptide fragments to display low IgEbinding activity may be due to the fact that the contiguous overlappingpeptide fragments are linear and are unable to cross-link with IgEantibodies.

One important application of the invention is to the problem ofallergies to foods or materials in the surroundings. Millions ofindividuals are subjected to severe symptomatology in response tootherwise harmless components of the environment, for example, ragweedor other pollens. The method of the invention can prevent or diminishthis immune response which results in widespread discomfort.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention. As used herein and in theclaims, the singular forms “a,” “and” and “the” include plural referentsunless the context clearly dictates otherwise.

The terms “human leukocyte antigen” and HLA” is here defined as agenetic fingerprint on white blood cells and platelets, composed ofproteins that play a critical role in activating the body's immunesystem to respond to foreign organisms.

The term “plurality of contiguous overlapping peptide fragments (OPF)”is here defined as at least one, but most likely two, three, four, orfive, contiguous overlapping peptide fragments. For example, theschematic below shows an example of a plurality of contiguousoverlapping peptide fragments, if the alphabet was a 26 residue peptide,and the plurality contained four overlapping peptides: OPF₁₋₆, OPF₄₋₁₅,OPF₁₃₋₂₂ and OPF₂₀₋₂₆:

ABCDEF = OPF₁₋₆    DEFGHIJKLMNO = OPF₄₋₁₅             MNOPQRSTUV =OPF₁₃₋₂₂                    TUVWXYZ = OPF₂₀₋₂₆

The term “hypersensitive” is here defined as abnormally susceptiblephysiologically to a specific agent via IgE-mediated mechanisms (as anantigen or drug). Such antigen is in the present specification andclaims called an allergen.

The term “hyposensitive” is here defined as not being sensitive to aspecific agent (as an antigen or drug). Such antigen is in the presentspecification and claims called an allergen.

The terms “desensitize”, “immunological tolerance” or “tolerance” arehere defined as to make (a sensitized or hypersensitive individual)insensitive or nonreactive to a sensitizing agent (as an antigen ordrug) by a reduction in immunological reactivity of a host towardsspecific tolerated antigen(s). Such antigen is in the presentspecification and claims called an allergen.

The term “positive-control” is here defined as a native allergen thatwhen applied to the skin will produce a positive reaction i.e. a redarea, the flare and a raised spot, the wheal, at the test site if IgEantibody is present. Apart native allergens, examples ofpositive-controls include pharmacological agents such as, but notlimited to, histamine. The optimal positive-control is the allergenitself in its native confirmation.

The term “negative-control” is here defined as a composition that whenapplied to the skin, should not produce, at 15 minutes, a response witha flare >5 mm when the injected volume of solution (50 μl) producesspontaneously a papule of 5 mm. Negative-controls include OPF diluent,albumin solution or saline (salt-water) solution.

The term “papule” is here defined as a small circumscribed, superficial,solid elevation of the skin. When related to allergens, it is usuallymeasured by a wheal and flare reaction which is an outward spreadingzone of reddening flare followed rapidly by a wheal (swelling) at thesite of introduction of the allergen.

The term “erythema” is here defined as redness of the skin produced bycongestion of the capillaries, which may result from a variety ofcauses.

The term “isolated” or “purified” peptide fragments or biologicallyactive portion thereof is substantially free of material (e.g., other,contaminating proteins) from the cell suspension, tissue source, orserum preparation from which the allergen peptide fragments are derived,or substantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of othermaterial” includes preparations of the allergen-derived peptidefragments in which the peptide fragments are separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the peptide fragments having less thanabout 30% (by dry weight) of non-allergen protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-allergen protein, still more preferably less than about 10%of non-allergen protein, and most preferably less than about 5%non-allergen protein. When the allergen-derived peptide fragments arerecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the overlapping peptides preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the allergen-derived peptidefragments in which the peptide fragments are separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of theallergen-derived peptide fragments having less than about 30% (by dryweight) of chemical precursors or non-allergen chemicals, morepreferably less than about 20% chemical precursors or non-allergenchemicals, still more preferably less than about 10% chemical precursorsor non-allergen chemicals, and most preferably less than about 5%chemical precursors or non-allergen chemicals.

Manipulations of the sequences included within the scope of theinvention may be made at the peptide level. Included within the scope ofthe present invention are peptide fragments (derivative or analogthereof) that are modified during or after translation or synthesis(e.g., by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, andthe like). Any of the numerous chemical modification methods knownwithin the art may be utilized including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH.sub.4, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc. In a specificembodiment, sequences of a peptide are modified to include a fluorescentlabel Allergen-derived peptide fragments, analogs, derivatives, andvariants thereof can be chemically synthesized. For example, a peptidefragment corresponding to a portion of an allergen protein that includesa desired domain or that mediates a desired activity in vitro, may besynthesized by use of a peptide synthesizer. The amino acid sequence ofa protein isolated from the natural source, may be determined, e.g., bydirect sequencing of the isolated protein. The protein may also beanalyzed by hydrophilicity analysis (see, Hopp and Woods, PNAS USA78:3824, 1981) which can be used to identify the hydrophobic andhydrophilic regions of the protein, thus aiding in the design ofpeptides for experimental manipulation, such as in binding experiments,antibody synthesis, etc. Secondary structural analysis may also beperformed to identify regions of a peptide that adopt specificstructural motifs. (See, Chou and Fasman, Biochem, 13:222, 1974).Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies, canbe accomplished using computer software programs available in the art.Other methods of structural analysis including, but not limited to,X-ray crystallography (see, Engstrom Biochem Exp Biol 11:7, 1974); massspectroscopy and gas chromatography (see, Methods in Protein Science J.Wiley and Sons, New York, N.Y. 1997); computer modeling (see, Fletterickand Zoller, eds., 1986, Computer Graphics and Molecular Modeling, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); optical rotary dispersion(ORD) and circular dichroism (CD) may also be used. For example,measurement of circular dichroism may be used to determine the linearityof candidate peptides for use as COPs.

The peptide fragments, derivatives and other variants described herein,can be modified. Thus, the invention includes, e.g., myristylated,glycosylated, palmitoylated and phosphorylated peptides and theirderivatives.

Conservative amino acid substitutions can be made in the peptidefragments at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in apeptide fragment with a conservative amino acid substitution a predictednon-essential amino acid residue in the allergen-derived fragment ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of the allergen coding sequence,to identify mutants that retain T cell stimulating activity but havelower or reduced/weak levels of IgE stimulating activity.

In some embodiments, a mutant allergen peptide fragment can be assayedfor (1) the ability to stimulate or induce T cell proliferation or (2)the ability, or lack of, to bind IgE antibodies from, e.g., the sera ofan individual hypersensitive to the allergen. The terms “stimulate” or“induce” are used interchangeably herein.

A peptide fragment or combination of overlapping peptide fragmentsderived from a protein allergen, can be tested to determine whether thepeptide will produce local or systemic symptoms that are related to aType I reaction. This reaction involves the interaction of antigen withantibody of the immunoglobulin class IgE, which attaches to the hostcells in the skin and other tissues (mast cells, basophils, platelets,and eosinophils). An antigen encounter results in release of the cellcontents, including active molecules such as histamine, heparin,serotonin, and other vasoactive substances, producing local or systemicsymptoms that are manifest within minutes to a few hours followingantigen-IgE interaction. IgE binding activity of candidate COPs can alsobe measured by means of ELISA assays using IgEs specific for selectedpolypeptide allergens having less than a selected maximum bindingaffinity. According to one aspect of the invention, ELISA assays may beconducted on candidate COPS wherein COPs are selected which have abinding activity for IgE's reactive with the selected polypeptideallergen which is less than three times the standard deviation of anegative control in a conventional ELISA assay.

T cell stimulating activity can be tested by culturing T cells obtainedfrom an individual sensitive to the allergen proteins and variantsdescribed herein (i.e., an individual who has an immune response to theprotein allergen or protein antigen) with an allergen protein or variantand determining the presence or absence of proliferation by the T cellsin response to the peptide as measured by, for example, incorporation oftritiated thymidine. Stimulation indices for responses by T cells topeptides useful in methods of the invention can be calculated as themaximum counts per minute (cpm) incorporated in response to the peptidedivided by the cpm of the control medium. For example, a peptide derivedfrom a protein allergen may have a stimulation index of about 2.0. Astimulation index of at least 2.0 is generally considered positive forpurposes of defining peptides useful as immunotherapeutic agents.Preferred peptides or fragments or combinations of overlapping fragmentshave a stimulation index of at least 2.5, more preferably at least 3.5and most preferably at least 5.0.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., percent homologyequals the number of identical positions divided by the total number ofpositions times 100).

The invention also provides specific allergen chimeric or fusionproteins. As used herein, a specific allergen “chimeric protein” or“fusion protein” comprises, an allergen polypeptide operatively linkedto a non-allergen polypeptide. An “allergen polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a specificallergen, whereas a “non-allergen polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to a protein which is notsubstantially homologous to the specific allergen, e.g., a protein whichis different from the allergen and which is derived from the same or adifferent organism. Within a specific allergen fusion protein theallergen polypeptide can correspond to all or a portion of a specificallergen protein. In a preferred embodiment, a specific allergen fusionprotein comprises at least one biologically active portion of thespecific allergen. The non-allergen polypeptide can be fused to theN-terminus or C-terminus of the allergen polypeptide.

Allergen Based Compositions

The contiguous overlapping allergen peptide fragments (also referred toherein as “active compounds”) of the invention can be incorporated intocompositions suitable for administration. Such compositions typicallyinclude the contiguous overlapping peptide fragments and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to a carrier that does not cause an allergicreaction or other untoward effect in subjects to whom it isadministered. Suitable pharmaceutically acceptable carriers include, forexample, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol, or the like, and combinations thereof. In addition, if desired,the composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, and/or pH buffering agents whichenhance the effectiveness of the vaccine. Attention is directed toRemington's Pharmaceutical Science by E. W. Martin. Immunostimulatoryadjuvants are predominantly derived from pathogens, e.g.,lipopolysaccharide (LPS) and monophosphoryl lipid A (MPL), whichactivate cells of the immune system. Bacterial CpG motifs in DNA havedirect immunostimulatory effects on immune cells in vitro, theimmunostimulatory effect is due to the presence of unmethylated CpGdinucleotides, which are under-represented and are methylated invertebrate DNA. Unmethylated CpGs in the context of selective flankingsequences are thought to be recognized by cells of the immune system toallow discrimination of pathogen-derived DNA from self DNA. CpG motifsare most potent for the induction of Th1 responses, mainly throughstimulating TNFβ, IL-1, IL-6 and IL-12, and through the expression ofco-stimulatory molecules. CpGs also appear to have significant potentialas mucosally administered adjuvants. Importantly, CpGs also appear tohave significant potential for the modulation of existing immuneresponses, which may be useful in various clinical settings, includingallergies. (See for example, O'Hagan et al., Biomolecular Engineering,18:69-85, 2001; Singh and O'Hagan, Nature Biotechnology, 17:1075-1081,1999).

The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. As used herein, the phrases‘composition’ and ‘therapeutic composition’ are interchangeable.

Compositions containing the contiguous overlapping allergen peptidefragments, or variants thereof can be administered to a patient (such asa human) sensitive to the specific allergen in a form which results in adecrease in the T cell response of the mammal upon subsequent exposureto the protein allergen. As used herein, a decrease or modification ofthe T cell response of a mammal sensitive to a protein allergen isdefined as non-responsiveness or diminution in symptoms to the proteinallergen in the patient, as determined by standard clinical procedures(see, Varney et al., British Medical Journal, 302: 265, 1990), includingdiminution in allergen induced asthmatic conditions. As referred toherein, a diminution in symptoms to an allergen includes any reductionin the allergic response of a patient, such as a human, to the allergenfollowing a treatment regimen with a composition as described herein.This diminution in symptoms may be determined subjectively in a human(e.g., the patient feels more comfortable upon exposure to theallergen), or clinically, such as with a standard skin test orprovocation assay.

In addition, administration of the above-described contiguousoverlapping allergen peptide fragments or their variants may result inlower levels of IgE stimulation activity. Preferably, administrationresults in weak IgE stimulating activity. More preferably,administration results in zero IgE stimulating activity. As used herein,weak IgE stimulating activity refers to IgE production and/orcross-linking that is less than the amount of IgE production and/or IL-4production stimulated by the whole protein allergen.

Administration of the compositions of the present invention todesensitize or tolerize an individual to a protein allergen or otherprotein antigen can be carried out using procedures, at dosages and forperiods of time effective to reduce sensitivity (i.e., to reduce theallergic response) of the individual to a protein allergen or otherprotein antigen. Effective amounts of the compositions will varyaccording to factors such as the degree of sensitivity of the individualto the protein allergen, the age, sex, and weight of the individual, andthe ability of the peptide(s) to elicit a tollerogenic response in theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

A composition of the invention is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., skin prick, intravenous, intradermal,subcutaneous, oral, nasal, mucosal (e.g., inhalation), transdermal(topical), transmucosal, lymph node and rectal administration. Solutionsor suspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of toxicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Administration, e.g., subcutaneous administration, of anallergen-derived overlapping peptide or variant peptides as describedherein to a patient, such as a human, can tolerize or anergizeappropriate T cell subpopulations such that they become unresponsive tothe protein allergen and do not participate in stimulating an immuneresponse upon subsequent exposure. In addition, administration of such apeptide may modify the lymphokine secretion profile as compared withexposure to the naturally-occurring protein allergen or portion thereof(e.g., result in a decrease of IL-4 and/or an increase in IL-10, TGFβ,and IFN-γ). Furthermore, exposure to the peptide may influence T cellsubpopulations which normally participate in the response to theallergen such that these T cells, when re-exposed to the nativeallergen, are secreting high levels of IL-10, TGFβ, or IFN-γ, instead ofhigh levels of IL-4 or IL-5. This immune deviation of T cellsubpopulations may ameliorate or reduce the ability of an individual'simmune system to stimulate the usual immune response at the site ofnormal exposure to the allergen, resulting in a diminution in allergicsymptoms.

Compositions suitable for injectable use include sterile aqueoussolutions (where the peptides or protein are water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., overlapping peptide fragments) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The compositions can be included in a container, pack, or dispensertogether with instructions for administration.

It is also possible to modify the structure of peptides useful inmethods of the invention for such purposes as increasing solubility,enhancing therapeutic or preventive efficacy, or stability (e.g., shelflife ex vivo, and resistance to proteolytic degradation in vivo). Amodified peptide can be produced in which the amino acid sequence hasbeen altered, such as by amino acid substitution, deletion, or addition,to modify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose. For example, the aminoacid residues essential to T cell epitope function can be determinedusing known techniques (e.g., substitution of each residue anddetermination of presence or absence of T cell reactivity). Thoseresidues shown to be essential can be modified (e.g., replaced byanother amino acid whose presence is shown to enhance T cellreactivity), as can those which are not required for T cell reactivity(e.g., by being replaced by another amino acid whose incorporationenhances T cell reactivity but does not diminish binding to relevant MHCmolecules). Another example of a modification of peptides issubstitution of cysteine residues preferably with alanine, oralternatively with serine or threonine to minimize dimerization viadisulfide linkages.

In order to enhance stability and/or reactivity, peptides can also bemodified to incorporate one or more polymorphisms in the amino acidsequence of a protein allergen resulting from natural allelic variation.Additionally, D-amino acids, non-natural amino acids or non-amino acidanalogues can be substituted or added to produce a modified syntheticpeptide within the scope of this invention.

In some embodiments, the peptides can be synthesized as retro-inversopeptides. (See Sela and Zisman, FASEB J. 11:449, 1997). Evolution hasensured the almost exclusive occurrence of L-amino acids in naturallyoccurring proteins. Virtually all proteases therefore cleave peptidebonds between adjacent L-amino acids; thus, artificial proteins orpeptides composed of D-amino acids are largely resistant to proteolyticbreakdown. This resistance has been attractive to drug designers, butthe exclusivity of biological systems for proteins made of L-amino acidsmeans that such proteins cannot interact with the mirror image surfaceformed by enantiomeric proteins. Thus, an all D-amino acid proteinusually has no biological effect or activity.

Linear modified retro-peptide structures have been studied for a longtime (See Goodman et al., Accounts of Chemical Research, 12:1-7, 1979)and the term “retro-isomer” was designated to include an isomer in whichthe direction of the sequence is reversed compared with the parentpeptide. By “retro-inverso isomer” is meant an isomer of a linearpeptide in which the direction of the sequence is reversed and thechirality of each amino acid residue is inverted; thus, there can be noend-group complementarity.

More recently, Jameson et al. engineered an analogue of the hairpin loopof the CD4 receptor by combining these two properties: reverse synthesisand a change in chirality. See Jameson et al., Nature 368:744-746, 1994and Brady et al., Nature, 368:692-693, 1994. The net result of combiningD-enantiomers and reverse synthesis is that the positions of carbonyland amino groups in each amide bond are exchanged, while the position ofthe side-chain groups at each alpha carbon is preserved. Jameson et al.demonstrated an increase in biological activity for their reverse Dpeptide, which contrasts to the limited activity in vivo of itsconventional all-L enantiomer (due to its susceptibility toproteolysis).

A partially modified retro-inverso pseudopeptide has been reported foruse as a non-natural ligand for the human class I histocompatibilitymolecule, HLA-A2. (See Guichard et al., Med. Chem. 39:2030-2039, 1996).Such non-natural ligands had increased stability and high MHC-bindingcapacity.

Retro-inverso peptides are prepared for peptides of known sequence inthe following manner. A peptide having a known sequence (e.g., a tumorantigen peptide) is selected as a model peptide for designing andsynthesizing a retro-inverso peptide analog. The analog is synthesizedusing D-amino acids by attaching the amino acids in a peptide chain suchthat the sequence of amino acids in the retro-inverso peptide analog isexactly opposite of that in the selected peptide which serves as themodel. To illustrate, if the peptide model is a peptide formed ofL-amino acids having the sequence ABC, the retro-inverso peptide analogformed of D-amino acids would have the sequence CBA. The procedures forsynthesizing a chain of D-amino acids to form the retro-inverso peptidesare known in the art and are illustrated in the above-noted references.

Since an inherent problem with native peptides is degradation by naturalproteases, the peptides of the invention may be prepared to include the“retro-inverso isomer” of the desired peptide. Protecting the peptidefrom natural proteolysis should therefore increase the effectiveness ofthe specific heterobivalent or heteromultivalent compound.

A higher biological activity is predicted for the retro-inversocontaining peptide when compared to the non-retro-inverso containinganalog owing to protection from degradation by native proteinases.

Furthermore, peptides can be modified to produce a peptide-PEGconjugate. Modifications of peptides can also includereduction/alkylation (Tarr in: Methods of Protein Microcharacterization,J. E. Silver, ed. Humana Press, Clifton, N.J., pp 155-194, 1986);acylation (Tarr, supra); esterification (Tarr, supra); chemical couplingto an appropriate carrier (Mishell and Shiigi, eds., Selected Methods inCellular Immunology, WH Freeman, San Francisco, Calif.; U.S. Pat. No.4,939,239, 1980); or mild formalin treatment (Marsh InternationalArchives of Allergy and Applied Immunology, 41:199, 1971).

To facilitate purification and potentially increase solubility ofpeptides, it is possible to add reporter group(s) to the peptidebackbone. For example, poly-histidine can be added to a peptide topurify the peptide on immobilized metal ion affinity chromatography.(See Hochuli et al., Bio/Technology, 6:1321, 1988). In addition,specific endoprotease cleavage sites can be introduced, if desired,between a reporter group and amino acid sequences of a peptide tofacilitate isolation of peptides free of irrelevant sequences. In orderto successfully desensitize an individual to a protein antigen, it maybe necessary to increase the solubility of a peptide by addingfunctional groups to the peptide or by not including hydrophobic T cellepitopes or regions containing hydrophobic epitopes in the peptide.

To aid proper antigen processing of T cell epitopes within a peptide,canonical protease sensitive sites can be recombinantly or syntheticallyengineered between regions, each comprising at least one T cell epitope.For example, charged amino acid pairs, such as KK or RR, can beintroduced between regions within a peptide during synthesis.

The invention further encompasses at least one therapeutic compositionuseful in treating a condition which involves an immune response to aprotein antigen (e.g., an allergen, an autoantigen, etc.) comprising atleast one peptide having a sufficient percentage of the T cell epitopesof the protein antigen such that in a substantial percentage of apopulation of individuals sensitive to the protein antigen, the responseof such individuals to the protein antigen is substantially diminished,with the provision that the at least one peptide does not comprise theentire protein antigen.

Bee Venom Allergens:

Bee venom (BV) is a complex mixture of antigens that can include one ormore toxic polypeptides. Many of these polypeptides are hypersensitizingagents and can additionally have hemolytic or neurotoxic effects.

Approximately 3% of the general population are hypersensitive to BVpolypeptides. IgE antibodies from BV hypersensitive individualsrecognize several BV toxic polypeptides. BV polypeptides, often referredto as allergens, recognized by IgE in BV hypersentive individuals caninclude, e.g., phospholipase A₂ (PLA₂), acid phosphatase, hyaluronidase,allergen C, and other, high molecular weight (MW) proteins.

BV hypersensitive individuals can be at high risk of an adverse reactionto a bee sting. One recognized method for preventing or minimizingserious adverse reactions resulting from a bee sting is to desensitizethe individual to the allergens present in BV. This protection can beinduced by a process termed venom immunotherapy (VIT).

Conventional VIT based on a standardized preparation of bee venomallergens provides complete protection in at least 80% of patients aftera 3-5 year desensitization. (See Kämmerer et al., Clin. Experiment.Allergy. 27:1016-1026, 1997).

Birch Pollen Allergens:

Birch pollen is a major source of type I allergies observed in earlyspring. An estimated 100 million individuals suffer from birch pollenallergy. Cross-linking of two IgE receptors on the surface of mast cellsand basophilic leucocytes, by allergen binding, initiates the release ofa number of physiologically active substances such as histamine, PAF(platelet activating factor), heparin, chemotactic factors foreosinophilic and neutrophilic granulocytes, leucotrienes, prostaglandinsand thromboxanes. It is these mediators which cause the direct symptomsof IgE-mediated allergic reactions (Type I hypersensitivity).

Bet v 1, the major birch pollen allergen, is composed of 160 amino acidresidues with a molecular weight of approximately 17 kDa. To date,eleven Bet v 1 protein sequence isoforms have been identified, withamino acid identities ranging from 84.4% ( 25/160 amino acid exchanges)to 99.4% (a single amino acid exchange). (See, Swoboda et al., J. Biol.Chem. 270(6):2607. 1995). Major three-dimensional structural features ofBet v 1 include a seven-stranded antiparallel beta-sheet that wrapsaround a long C-terminal alpha-helix, thereby forming a large cavity inthe interior of the protein.

Birch pollen profilin, Bet v 2, is composed of 133 amino acid residueswith a molecular weight of approximately 15 kDa. It is a structurallywell conserved actin- and phosphoinositide-binding protein and across-reactive allergen. Structural features include three α-helices andseven β-strands, as determined by NMR.

When peptides derived from birch pollen proteins or variants are used totolerize an individual sensitive to a protein allergen, the peptide ispreferably derived from a protein allergen of the genus Betulaverrucosa. The immunogenic features of rBet v 1 fragments/variants havebeen shown. (See, Vrtala et al., J. Immunol. 165:6653, 2000; vanHage-Hamsten et al., J. Allergy Clin. Immunol. 104(5):969, 1999; Vrtalaet al., Int. Arch Allergy Immun. 113:246, 1997; and Wiedermann et al.,Int. Arch. Allergy Immun. 126:68 2001).

Dust Mite Allergens:

The dust mite (DM) is a common cause of allergic rhinitis and asthma. Adust mite is a microscopic, eight-legged insect. More than 100,000 dustmites can be in a single gram of dust. People are not allergic to thedust mite itself, but to dust mite feces. Dust mites eat the microscopicskin dander found on people and animals, and then leave droppings. Eachdust mite can produce approximately 20 droppings each day. Dust mite arefound on people, animals and on almost every surface in homes, includingcarpet, upholstered furniture, mattresses and box springs, sheets andblankets, pillows and stuffed animals. When dead dust mites and dustmite droppings become airborne and are inhaled, they may produce anallergic reaction.

Two species of the mite genus Dermatophagoides, D. pteronyssinus and D.farinae, are important sources of house dust allergens. Two groups ofmajor allergens, Der 1 (Der p 1 and DER f 1) and Der 2 (Der p 2 and Derf 2), have been purified from these Dermatophagoides species.

Sequences and Corresponding SEQ ID Numbers:

The sequences and corresponding SEQ ID NOs discussed herein include thefollowing:

SEQ ID NO:1 PLA₂ fragment amino acid sequence (60 aa)

SEQ ID NO:2 PLA₂ fragment amino acid sequence (53 aa)

SEQ ID NO:3 PLA₂ fragment amino acid sequence (45 aa)

SEQ ID NO:4 PLA₂ amino acid sequence (134 aa)

SEQ ID NO:5 Bet v 1 fragment amino acid sequence (90 aa)

SEQ ID NO:6 Bet v 1 fragment amino acid sequence (80 aa)

SEQ ID NO:7 Bet v 1 amino acid sequence (160 aa)

SEQ ID NO:8 Bet v 2 fragment amino acid sequence (70 aa)

SEQ ID NO:9 Bet v 2 fragment amino acid sequence (73 aa)

SEQ ID NO:10 Bet v 2 amino acid sequence (133 aa)

SEQ ID NO:11 Der p 1 fragment amino acid sequence (81 aa)

SEQ ID NO:12 Der p 1 fragment amino acid sequence (86 aa)

SEQ ID NO:13 Der p 1 fragment amino acid sequence (86 aa)

SEQ ID NO:14 Der p 1 amino acid sequence (212 aa)

SEQ ID NO:15 Der p 2 fragment amino acid sequence (73 aa)

SEQ ID NO:16 Der p 2 fragment amino acid sequence (73 aa)

SEQ ID NO:17 Der p 2 amino acid sequence (136 aa)

TABLE 1 Amino Acid Sequences of the Invention Amino Acid SequenceSEQ ID NO IIYPGTLWCGHGNKSSGPNELGRFKHTDACCRTHDMCPDVMSAGESKHGLTNTASHTRLS(SEQ ID NO: 1) KHGLTNTASHTRLSCDCDDKFYDCLKNSADTISSYFVGKMYFNLIDTKCYKLE(SEQ ID NO: 2) LIDTKCYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKY(SEQ ID NO: 3)IIYPGTLWCGHGNKSSGPNELGRFKHTDACCRTHDMCPDVMSAGESKHGLTNTASHTRLS(SEQ ID NO: 4)CDCDDKFYDCLKNSADTISSYFVGKMYFNLIDTKCYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKYMGVFNYETEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFP(SEQ ID NO: 5)EGFPFKYVKDRVDEVDHTNFKYNYSVIEGGHPVTGCGERTEGRCLHYTVDKSKPKVYQWF DLRKYKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEM(SEQ ID NO: 6) GETLLRAVESYLLAHSDAYNMGVFNYETEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFP(SEQ ID NO: 7)EGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNMSWQTYVDEHLMSDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPG(SEQ ID NO: 8) HLAPTGLHLGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQSNMV(SEQ ID NO: 9) VERLGDYLIDQGLMSWQTYVDEHLMSDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPG(SEQ ID NO: 10)HLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQSNMVVERLGDYLIDQGLTNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQ(SEQ ID NO: 11) ELVDCASQHGCHGDTIPRGIESQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNK(SEQ ID NO: 12) IREALAQTHSAIAVIIGIKDLDAFRHAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNW(SEQ ID NO: 13) GDNGYGYFAANIDLMMIEEYPYVVILTNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQ(SEQ ID NO: 14)ELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVILDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFEANQNTKTAKIEIKASIDG(SEQ ID NO: 15) LEVDVPGIDPNASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGV(SEQ ID NO: 16) LACAIATHAKIRDLVAAVARDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFEANQNTKTAKIE(SEQ ID NO: 17)IKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRD

Chimeric Allergens

The present invention further provides compositions and kits fordiagnostic use that are comprised of one or more containers containing achimeric allergen protein and contiguous overlapping peptide fragments.The chimeric allergen protein and peptide fragments are comprised ofpeptide fragments from different allergens (e.g. one or more fromallergen one with one or more from allergen two from the same class ofallergen (e.g. bee venom, birch pollen, dust mite, etc.)). The kit may,optionally, further comprise a series of compositions of knownconcentration, a positive-control and a negative-control in theaforementioned assays.

In a preferred embodiment the chimeric protein comprises peptidefragments within a specified allergen class. For example, chimericproteins comprising Bet v 1 (SEQ ID NO:5 and 6) and Bet v 2 (SEQ ID NO:8and 9) peptide fragments or Der p 1 (SEQ ID NO:11-13) and Der p 2 (SEQID NO:15 and 16). These peptide fragments would be contiguous, howeverthe fragments can be distant from each other and in various orientationsand may include overlapping peptides.

For example, the schematic below shows an example of overlapping peptidefragments:

ABCDEF = OPF (1 fragment 1) DEFGHI = OPF (1 fragment 2) 123456 = OPF (2fragment 1) 456789 = OPF (2 fragment 2)

which can be used to generate overlapping chimeric peptide fragments,for example:

ABCDEF123456 = OPF (Chimeric fragment 1) 456789DEFGHI = OPF (Chimericfragment 2) OR 123456ABCDEF = OPF (Chimeric fragment 3) DEFGHI456789 =OPF (Chimeric fragment 4)

In another embodiment, the chimeric protein comprises peptide fragmentsfrom different allergen classes. For example, chimeric proteinscomprising PLA₂ (SEQ ID NO:1-3) and Bet v 1 (SEQ ID NO:5 and 6) or Bet v2 (SEQ ID NO:8 and 9) peptide fragments or chimeric proteins comprisingPLA₂ (SEQ ID NO:1-3) and Der p 1 (SEQ ID NO:11-13) or Der p 2 (SEQ IDNO:15 and 16). Chimeric peptide fragments from different allergens areuseful in diagnosing patients with different allergies. For example,chimeric proteins comprising PLA₂ and Bet v 1 or Bet v 2 would beapplicable to patients allergic to both bee venom and birch pollen.

Any chimeric protein, or fragment or combinations thereof, comprisingSEQ ID NOs: 1-3, 5, 6, 8, 9, 11-13, 15 and 16 is included in the presentinvention. Preferred chimeric peptide fragments are listed in Table 2.For example, SEQ ID NO:18 comprises (in linear arrangement) SEQ ID NOs:5and 8; SEQ ID NO:19 comprises SEQ ID NOs:9 and 6; SEQ ID NO:20 comprisesSEQ ID NOs:8 and 5; SEQ ID NO:21 comprises SEQ ID NOs:6 and 9; SEQ IDNO:22 comprises SEQ ID NOs:15 and 11 and SEQ ID NO:23 comprises SEQ IDNOs:13 and 16.

TABLE 2 CHIMERIC AMINO ACID SEQUENCES Amino Acid Sequence SEQ ID NOMGVFNYETEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIK (SEQ ID NO: 18)KISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGMSWQTYVDEHLMSDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKDFEEPGHLAPTGLHLGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPG (SEQ ID NO: 19)QSNMVVERLGDYLIDQGLKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNMSWQTYVDEHLMSDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGIMKD (SEQ ID NO: 20)FEEPGHLAPTGLHLGMGVFNYETEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVK (SEQ ID NO: 21)ASKEMGETLLRAVESYLLAHSDAYNHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGITIKKTGQALVFGIYEEPVTPGQSNMVVERLGDYLIDQGLDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFEANQNTKTAKIEIK (SEQ ID NO: 22)ASIDGLEVDVPGIDPNATNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNS (SEQ ID NO: 23)WDTNWGDNGYGYFAANIDLMMIEEYPYVVILSIDGLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRD

Kits Including Allergens

The present invention additionally provides kits for diagnostic use thatare comprised of one or more containers containing a specific allergenprotein and contiguous overlapping peptide fragments. The kit may,optionally, further comprise a series of compositions of knownconcentration, a positive-control and a negative-control in theaforementioned assays.

Allergies to various allergens can be treated with the compositions andmethods of the invention. Examples of allergens include, but are notlimited to:

The following Examples are presented in order to more fully illustratethe preferred embodiments of the invention. These Examples should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLES Example 1 Bee Venom Specific T Cell Tolerance Induction withAllergen-Derived Overlapping Peptide Fragments

This study was designed to evaluate the safety and immunogenicity of anallergen-derived overlapping peptide fragment (OPF) immunotherapy.

Materials and Methods

Patients: Sixteen bee venom (BV) hypersensitive patients were recruitedfrom the Outpatient Clinic of the Division of Allergy and Immunology,Lausanne, Switzerland (9 males/7 females). Criteria for enrollment weregrade I to IV systemic hypersensitivity reaction to honey bee fieldsting (Müller J. Asthma Res. 3:331-333, 1996); positive anti-PLA₂ andanti-whole BV specific IgE (>0.35 kU/1 as titrated by CAP system,Pharmacia, Uppsala, Sweden, and by immunoblotting), positive immediateintradermal (ID) skin tests to phospholipase A₂ (PLA₂) and whole BV(presence of a wheal >5 mm with erythema at an allergen concentration≦0.1 μg/ml) and negative ID test to individual OPF and OPF mixture (≦5mm wheal and flare reaction at peptide concentration >0.1 μg/ml).

Peptide synthesis and purification: Three overlapping peptide fragmentsOPF₁₋₆₀ (SEQ ID NO:1), OPF₄₇₋₉₉ (SEQ ID NO:2) and OPF₉₀₋₁₃₄ (SEQ IDNO:3) mapping the entire 134 amino acids of PLA₂ (SEQ ID NO: 4) fromApis mellifera were synthesized on an Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer, Foster City, Calif.) and purified asdescribed in Roggero et al., FEBS Lett. 408:285-288, 1997. AnalyticalHPLC and mass spectrometry were used to assess the purity of eachpeptide (>80%), which were readily soluble in PBS. On the day ofinjection, the peptide mixture was reconstituted in an 0.3 mg/ml albuminsolution (containing 4 mg/ml of phenol) (ALK/Abello, Horsholm, Denmark)and injected subcutaneously in the deltoid area.

Skin testing: ID tests with BV, PLA₂ and peptides were performed asdescribed in Müller et al., Allergy 48(14):37-46, 1993. Concentrationstested ranged from 10⁻³ μg/ml to 1 μg/ml (10-fold dilution series). AnID test result was considered positive when a wheal reaction superior to5 mm (for BV, PLA₂ and peptides) in diameter and an erythema werepresent at a concentration ≦0.1 μg/ml. The 0.1 μg/ml concentration wasdefined as the end-point concentration (EPC), as higher concentrationsof BV and PLA₂ may induce non specific toxic reactions. See Müller etal., J. Allergy Clin. Immunol. 96:395-402, 1995.

Study design: The study was designed as a double blind, randomized,two-dose, placebo-controlled trial. At day 0, patients (n=9) from theOPF group were injected at 30 min interval with successively 0.1 μg, 1μg, 10 μg, 20 μg, 40 μg, 80 μg and 100 μg of each of the three OPFs(cumulative dose of 251.1 μg of each OPF within 3 h). Seven patientswere then injected at day 4, 7, 14, 42 and 70 with a maintenance dose of100 μg of each of the three OPFs. A maintenance dose of 300 μg of eachOPF was initially injected to two patients up to day 42. Patients fromthe control group (n=7) were injected with an equivalent volume ofpeptide diluent only (0.3 mg/ml albumin solution, containing 4 mg/ml ofphenol) (ALK/Abello, Horsholm, Denmark).

Reagents: Whole BV and PLA₂ were purchased from Latoxan (Rosans,France). For cell culture, PLA₂ was further purified by HPLC. Itscytotoxicity was inhibited by overnight reduction at 37° C. with a 100molar excess of dithiothreitol, followed by alkylation with a 1000 molarexcess of N-ethylmaleimide. PLA₂ was finally purified on a Sephadex G-25column (Pharmacia, Uppsala, Sweden). PMA and ionomycin were purchasedfrom Calbiochem, San Diego, Calif.

Proliferation assays: Blood was drawn immediately before each OPFinjection and peripheral blood mononuclear cells (PBMC) were isolatedfrom heparinized blood by density gradient centrifugation overFicoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Prior to³H-thymidine (Du Pont NEN Products Boston, Mass., USA) incorporation,PBMC (2×10⁵/well) from each donor were cultured for 6 days inoctoplicates in 96 well flat bottom plates (Costar Corning Inc., NewYork, N.Y.) in RPMI 1640 medium (Gibco, Basel, Switzerland) containing10% AB⁺ serum (Swiss Red Cross, Bern, Switzerland), 2 mM glutamine, 1%Na-pyruvate, 1% non-essential amino acids, 1% kanamycine (all fromGibco) with optimal concentration of OPFs (10 μg/ml) or PLA₂ (10 μg/ml).See Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103, 1997.

Short term T cell lines: T cell lines were derived from PBMC that wereisolated before each injection and stimulated in 24 well plates (Nunc)(10⁶ cells/well) with a mixture of the three OPFs (10 μg/ml) for 7 daysin supplemented 10% AB⁺ RPMI 1640 medium as described above. The shortterm T cell lines obtained were washed and restimulated for 24 h (forIL-4, IL-5, IL-13 and TGFβ secretion) or 48 h (for IFNγ and IL-10) withplastic crosslinked OKT3 (1 μg/ml) (see Jutel et al., Clin. Experiment.Allergy 25:1108-1117, 1995). Cell culture supernatants were collectedfor cytokine quantification and stored at −80° C.

Cytokine quantification: IL-4, IL-10 and IFNγ were titrated usingcommercially available ELISA kits (Mabtech AG, Nacka, Sweden, for IL-4,IL-10 and IFNγ □□ and R&DSystem for IL-5, IL-13 and TGFβ), according tomanufacturer's recommendations.

Quantification of specific serum IgE and IgG₄: Whole BV and anti-PLA₂specific IgE were quantified using the Phamarcia CAP SystemFluoroimmunoassay (Pharmacia Diagnostic AB, Uppsala, Sweden) asdescribed in Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997. For quantification of specific anti-PLA₂ IgG4, native PLA₂ (5μg/ml) was coated on 96 well plates (Maxisorb, Denmark) incarbonate/bicarbonate buffer pH 9.6, for 2 h at room temperature. Plateswere blocked with milk 5%/PBS/Tween 0.05%. Serial dilutions of sera in1% milk/Tween 0.05% were incubated for 1 h at room temperature. Plateswere washed thrice, incubated with horseradish peroxidase labeledanti-IgG4 mAb JDC-14 1/10,000 (Pharmingen, Hamburg, Germany), andrevealed in 3,3′, 5,5′-tetramethylbenzidine (TMB). Optical density wasdetermined at 450 nm on a microtiter plate analyzer (MR5000, DynatechLaboratories). Titers were reported to a standard serum and expressed asarbitrary standard units.

Immunoblotting and dot blot analysis: Anti-BV or -PLA₂ immunoblots wereprocessed as described in Kettner et al., Clin. Experiment. Allergy29:394-401, 1999. For dot blot analysis, 1 μg of whole BV, PLA₂, OPFs orhuman albumin was diluted ¼ in DMSO, spotted on PVDF membranes and driedfor 30 min. at 37° C. After blocking in non-fat milk 5%, further stepswere performed as described in Kettner et al., Clin. Experiment. Allergy29:394-401 1999. Dot densities were analyzed by scanning densitometryusing an Advanced American Biotechnology scanner, Fullerton, Calif.

Statistical analysis: Differences within and between groups wereevaluated by non-parametric ANOVA tests (Friedman or Kruskal-Wallis nonparametric test with multicomparison post-test, or Mann-Whitney testrespectively); or by Fisher's exact test (between group differences:responders versus non-responders, positive responses being defined as adoubling of day 0 value), using an Instat 3.0 software.

Results

Patients' data: Patients were randomly assigned to the OPF or control(albumin) groups. In the OPF group, mean age of patients was 39±14 yrs(5 males/4 females). One patient had a previous history of grade Ihypersensitivity to BV, 7 a grade III and one a grade IV according toMueller's classification. EPC for ID tests to BV was 10^(−1.7) μg/ml(geometric mean). Mean serum anti-BV specific IgE level was 21.5±33.9kU/l. In the control group, mean age was 40±10 yrs (4 males/3 females).One patient had previously developed a grade I hypersensitivity reactionto bee venom, three a grade II and three a grade III. EPC for ID teststo BV was 10^(−2.0) μg/ml (geometric mean). Mean serum anti-BV specificIgE level was 29.8±26.1 kU/l. There was no significant differencebetween groups at inclusion regarding sex, ages, severity of initialclinical reaction, anti-BV IgE and anti-PLA₂ specific IgE and IgG4antibody levels.

Overlapping peptide immunotherapy induces T cell anergy: In both groups,PBMC collected before each OPF or albumin injection were stimulated withthe three OPF mixture (10 μg/ml). As reported in Kämmerer et al., J.Allergy Clin. Immunol. 100:96-103, 1997, T cell proliferation inresponse to the three OPFs (expressed as the ratio of T cell response toPMA (100 ng/ml)/Ionomycine (1 μM) used as internal control) before thefirst injection at day 0 was low in either group all along the study,and persisted so in the control group (Friedman, p>0.05) (FIG. 1). Incontrast, there was a marked enhancement of T cell proliferation ratioin response to the three OPFs in the peptide group, which wassignificant both within (Friedman, p=0.035) and between groups(Mann-Whitney, day 14 and day 42, p<0.05). Proliferation ratio medianrose from 0.03 to 0.22 at day 14 to progressively decrease thereafter tothose obtained in the control group, demonstrating an active toleranceinduction. This pattern thus demonstrated that T cell toleranceoccurring after day 42 in the peptide group was preceded by a vigorousactivation phase peaking at day 14.

T cell cytokine production: PBMC collected before each injection werestimulated with a mixture of the three OPFs for 7 days, then activatedwith OKT3 (1 μg/ml) for 24 to 48 hr, following previously describedprotocols (Jutel et al., Clin. Experiment Allergy 25:1108-1117, 1995).IL-4 secretion by PBMCs maximally stimulated with OKT3 remained low inthe peptide group (FIG. 2A). A similar pattern was observed for IL-5 andIL-13 secretion. In contrast, we observed a striking enhancement of bothIFNγ and IL-10 secretion by OPF specific T cells, which reached a peakat day 42 of therapy (Kruskal-Wallis, p<0.018 and <0.012 respectively)(FIG. 2B, 2C). IL-10 and IFNγ secretion tended to decline towards day 80(non-significant). TGFβ secretion stayed at background level all alongthe trial. There was in contrast no change overtime in IL-4, IL-5,IL-10, IL-13, TGFβ and IFNγ production by PBMCs isolated from thecontrol group. These data were compatible with a TH0 to TH1 immunedeviation paralleled by an enhanced production of IL-10, a cytokine thatmay be involved in the active T cell tolerance induction observed (FIG.1). See Akdis et al., J. Clin. Invest. 102:98-106, 1998.

Specific anti-PLA2 serum IgE and IgG₄: Serum anti-PLA₂ IgE were measuredat screening visit, at days 14, 42 and 80 using a CAP assay. Though thedifference between the anti-PLA₂ IgE levels overtime in the peptideversus the control group indicated a trend towards higher IgE value inthe peptide group (Fisher's exact test, T14, T42, T80, p<0.03),comparison within the groups showed that there was no significantvariation of anti-PLA2 IgE levels overtime (Friedman, p>0.05) (FIG. 3A,B). In contrast, specific anti-PLA₂ IgG4 antibodies steadily increasedovertime within the peptide group to reach significance (Friedman,p<0.001) (FIG. 4A). Each point represents an individual value.Differences within the groups was statistically non-significant(Friedman p>0.05). Serum anti-PLA₂ IgG4 levels in the control group(FIG. 4B) remained constant all over the study and significantlydiffered from the peptide group (Fisher's exact test, T42, T70, T80,p<0.01).

Skin immediate reactivity to intradermal tests: At the screening visit,none of the patients in the OPF or in the control group developed animmediate allergic reaction to intradermal injection of any of the threeOPFs separately or as a mixture (EPC≧1 μg/ml) (FIG. 5 and FIG. 6A, 6C).Each point represents an individual value. Differences within groupswere examined by Friedmann non-parametric ANOVA test (p<0.001 forpeptide group, p>0.05 for control group), completed in panel A by amulticomparison post-test (p<0.01, p<0.05 and p<0.05 for day 0 vs day42, 70 and 80 respectively). At the end of the trial (day 70), none ofthe patients from the control group had EPC ≦0.1 μg/ml, whereas four outof the nine patients from the peptide group developed skin reactivity tothe OPF mixture at 0.1 μg/ml, considered as the lower limit ofpositively. At day 0, all patients in the OPF and control group hadpositive ID tests to native PLA₂ (FIG. 6B, 6D). At the end of the trial(day 70), in the peptide group (FIG. 6B), two patients increased theirEPC by two log₁₀, and two others by one log₁₀. A single patientdecreased his EPC to PLA₂ from 0.1 to 0.01 μg/ml, whereas four patientsdid not change their EPC to PLA₂. In the control group at day 70 (FIG.6B), two patients increased their EPC to PLA₂ by one log, one patientdecreased his EPC by one log and four did not modify their EPC. Thoughglobally those changes were non significant between the groups, the onlytwo patients who markedly enhanced their EPC to native PLA₂ (by twologs) were issued from the OPF group.

In vitro IgE binding to overlapping peptide fragments: In vitro specificIgE response to BV, native PLA₂ and each of the three OPFs was tested bydot assays at days 0, 7, 14, 42, 70 and 80 (FIG. 7). Though there was atrend to a modestly enhanced mean anti-whole BV and anti-native PLA₂ IgEbinding in the peptide group at day 14 and later, as compared to days 0and 7, there were no significant difference within and between thegroups (FIG. 7A, 7B). Similarly, there were no differences in IgEbinding to individual OPF within and between the groups (FIG. 7C, 7D,7E). Again, a non-significant trend towards enhanced IgE recognition ofpeptide OPF₉₀₋₁₃₄ was noted in the OPF group. Both in the control andpeptide groups, the C-terminal peptide OPF₉₀₋₁₃₄ was binding IgE at ahigher level followed by the N-terminal peptide OPF₁₋₆₀. IgE binding tothe internal peptide OPF₄₇₋₉₉ was undetectable. Intradermal test withnative PLA₂ only was positive.

Safety evaluation study: At day 0, despite the injection of sharplyincreasing OPF doses up to a cumulative dose of 250 μg of each peptidewithin 3.5 hrs (100 μg OPF group), none of the patients experiencedlocal or systemic reactions. In two patients, mild, late (>2 hrs) localreactions (erythema) occurred after peptide injection at day 14, 42 and70 to vanish after about an hour. In those two patients, after the lastinjection at day 70, hand palm pruritus and transient erythema of theupper part of the trunk occurred more than 3 h after OPF injection.There were no severe adverse events (life threatening reactions).

A maintenance dose of 300 μg OPF was initially injected to two patients.In one patient, the late occurrence (>2 hrs) of local skin reaction andupper trunk flush at day 42 led to the interruption of the treatment.The other patient, for safety reasons, was subsequently allocated to the100 μg OPF treatment group, though the 300 μg dosage was well tolerated.

Discussion

This study showed that a peptide based allergen immunotherapy using OPFsderived from PLA₂, a major BV allergen, was able to induce T cellanergy, immune deviation toward a Th1 type T cell cytokine response,enhanced IL-10 secretion and PLA₂ specific IgG4 production. OPFimmunotherapy was safe and did not induce severe systemic reactionsthough dose cumulation appeared to induce mild, non-immediate reactionsin two patients.

The fact that OPFs could be injected without any local or systemicadverse events at day 0, though cumulative doses of each peptide werereaching more than 250 μg (550 μg in the two patients injected with 300μg OPFs) demonstrates the high safety profile of OPFs. Mild localreactions (pruritus and erythema) occurred in only two patients at day14, 42 and 70 more than 120 min. after the injection and did not lastfor more than one hour. In the same patients, the ultimate peptideinjection led to late (>3 h) systemic reactions characterized by handpruritus and a flash of the upper trunk. This presentation is nottypical of anaphylaxis, since it occurred relatively late (>3 hrs) ascompared to usual anaphylactic reactions during conventionalimmunotherapy or rush protocols that are triggered within minutes. Thedelayed character of these reactions were suggestive of a late allergicreaction, as interpreted in previous allergen peptide trials (Norman etal., Am. J. Respir. Crit. Care Med. 154:1623-1628, 1996; Oldfield etal., J. Immunol. 167:1734-1739, 2001; and Haselden et al., J. Exp. Med.189:1885-1894, 1999) and may be related to the stimulation of specific Tcells to produce TH1 pro-inflammatory cytokines. These secondary eventsare dose-dependent (Oldfield et al., J. Immunol. 167:1734-1739, 2001),what certainly suggests a need to adapt the dose of OPFs in furtherclinical evaluations of OPF immunotherapy. Reactions were however allbenign and self-limited. No life-threatening reactions occurred.

In vitro dot blot assays, a non-significant trend toward an enhanced IgEbinding to native PLA₂, whole BV or peptides was apparent after thethird OPF injection. Taken together with the trend in serum anti-PLA₂IgE level increase, these data suggest that in OPF immunotherapy, as inconventional BV immunotherapy, an increase in allergen specific IgE mayoccur during the first weeks of treatment (Kämmerer et al., J. AllergyClin. Immunol. 100:96-103, 1995 and Müller Insect Sting Allergy:clinical picture, diagnosis and treatment. Stuggart, New York: GustavFischer Verlag, 1990). This increase may essentially reflect thetransient specific T cell activation observed during the first two weeksof therapy. IgE binding activity to peptides was clearly limited andplateaued after day 42. It was not reflected by in vivo skin testing atinitiation of the study. During the course of the trial, four patientsdeveloped mildly positive ID tests to OPFs at 0.1 μg/ml, whereas thefive others were still negative at 1 μg/ml. This difference wascertainly significant since in the control group none of the patientshad positive ID tests at 0.1 μg/ml concentration at the end of thetrial. The clinical significance of these positive ID tests is howeverdifficult to appreciate: the two patients who developed mild systemicreactions after day 70 injection were among those four patients.However, clinical tolerance to OPF injection was good in the two others.Longer term studies on larger study population will be necessary toassess the long term safety of OPF-based immunotherapy.

One of the most prominent results of this study was the induction of aprofound specific T cell hyporesponsiveness at day 80. If at thescreening visit, T cell proliferation in response to OPFs was low, whatessentially suggested a low number of BV specific T cell precursors, itpeaked at day 14 in the peptide group before progressing tohyporesponsiveness. Although previously shown in murine models (Tsitouraet al., J. Immunol. 163:2592-2600, 1999; Hoyne et al., Int. Immunol.8:335-342 1996; and Pape et al., J. Immunol. 160:4719-4729, 1998), theseresults demonstrate in humans that anergy induction was preceded by Tcell activation. This observation is in agreement with the recentdemonstration that the late asthmatic reaction induced by the firstadministration of allergen-derived T cell peptides in cat allergicasthmatics preceded the induction of antigen-hyporesponsiveness(Oldfield et al., J. Immunol. 167:1734-1739, 2001. The progressivedown-regulation of T cell response to OPFs was paralleled by enhancedIL-10 and IFN-γ secretion, peaking at day 42 to decrease thereafter. Thepattern of T cell proliferation overtime was suggestive of T cell anergyinduction. T cell clonal deletion may have also contributed to thephenomenon, especially with regard to the decrease in cytokine secretionoccurring late in the course of therapy. Interestingly, the peak ofIL-10 and IFNγ secretion occurred about 4 weeks after the maximal T cellproliferation, i.e. at a time when T cell anergy was established. Thissituation is not incompatible with T cell tolerance, since in vitroanergic CD4⁺ T cell clones are still able to differentiate into Th1-likeeffector cells, to participate in T-dependent IgG2a anti-haptenresponses and delayed-type hypersensitivity reactions (Malvey et al., J.Immunol. 161:2168-2177, 1998. Similarly, in allergy models to PLA₂ inmice, a persistence of a strong IFNγ production and anti-allergen IgG2aresponse despite tolerance induction by OPFs was shown (von Gamier etal., Eur. J. Immunol 30:1638-1645, 2000 and Astori et al., J. Immunol.165:3497-3505, 2000). IL-10 has been involved in T cell anergy inductionand appeared to be secreted by a sub-population of T lymphocytes able torepress other CD4⁺ T cell specific activity, the so-called Tr1 subset(Groux et al., Nature 389:737-742,1997 and Akdis et al., FASEB J.13:603-609, 1999). IL-10 has also prominent anti-inflammatory properties(de Waal Malefyt et al., J. Immunol. 150:4754-4765, 1993). Though byitself an immune deviation to a TH1 type cytokine production may bedeleterious (Hansen et al., J. Clin. Invest. 103:175-183, 1999), acombination of an anti-inflammatory cytokine such as IL-10 and IFNγ mayre-equilibrate a potentially detrimental cytokine secretion.

Specific anti-PLA₂ serum IgG4 response was significantly stimulated.Previously, a gradual rise in IgG4 during the incremental phase ofconventional immunotherapy has been demonstrated (Kämmerer et al., J.Allergy Clin. Immunol. 100:96-103, 1997 and Müller et al., Allergy44:412-418, 1989). Serum IgG4 levels may be predictive of effectiveprotection in response to immunotherapy (Urbanek et al., Clin. Allergy16:317-322, 1986 and Lesourd et al., J. Allergy Clin Immunol.83:563-571, 1989), though this concept may be controversial (Müller etal., Allergy 44:412-418, 1989). It was recently shown that IgE and IgG4levels obtained after 2 years of specific immunotherapy were specificand sensitive predictors of reactivity post hymenoptera venom challenge,a high IgG4 response being associated with protection and low IgG4levels with anaphylaxis (Ollert et al., J. Allergy Clin. Immunol.105:S59, Abstract 178, 2000). IgG4 may in part compete with IgE bindingon allergen and thus contribute to clinical protection (Schneider etal., J. Allergy Clin. Immunol 94:61-70, 1994).

This placebo-controlled trial demonstrated that an OPF-based allergenimmunotherapy was safe and able to induce specific T cellhyporesponsiveness, immune deviation toward TH1 cytokine secretion withparallel IL-10 secretion, and enhanced IgG4 production. As such, OPFimmunotherapy reproduces the pattern of cellular and humoral eventsobserved in rush and conventional immunotherapy without their inherentanaphylactic secondary events (Kämmerer et al., J. Allergy Clin.Immunol. 100:96-103, 1997; Akdis et al., J. Clim. Invest. 102:98-106,1998; Akdis et al., J Clin Invest 98:1676-83, 1996; and Jutel et al., J.Immunol. 154:4187-4194, 1995).

Example 2 Birch Pollen (Bet v 1) Specific T Cell Tolerance Inductionwith Allergen-Derived Overlapping Peptide Fragments

Materials and Methods

Patients: Patients eligible for this study include those with a historyof seasonal birch pollen allergy and with an SPT reaction ≧3+ comparedwith an albumin 10 mg/mL wheal and a minimal outcome of more than 3 mmwheal to commercial birch pollen extract.

Skin testing: Concentrations tested range from 10⁻³ μg/ml to 1 μg/ml(10-fold dilution series). An ID test result is considered positive whena wheal reaction superior to 5 mm (for birch pollen, Bet v 1 andpeptides) in diameter and an erythema are present at a concentration≦0.1 μg/ml.

Study design: The study is designed as a double blind, randomized,two-dose, placebo-controlled trial. At day 0, patients from the OPFgroup are injected at 30 min interval with successively 0.1 μg, 1 μg, 10μg, 20 μg, 40 μg, 80 μg and 100 μg of each of the two OPFs. Patients arethen injected at day 4, 7, 14, 42 and 70 with a maintenance dose of 100μg of each of the two OPFs. A maintenance dose of 300 μg of each OPF isinitially injected to two patients up to day 42. Patients from thecontrol group are injected with an equivalent volume of peptide diluentonly (0.3 mg/ml albumin solution, containing 4 mg/ml of phenol)(ALK/Abello, Horsholm, Denmark).

Peptide synthesis and purification: Two overlapping peptide fragmentsOPF₁₋₉₀ (SEQ ID NO:5) and OPF₈₀₋₁₆₀ (SEQ ID NO:6) mapping the entire 160amino acids of Bet v 1 (SEQ ID NO: 7) are synthesized on an AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.)and purified as described in Roggero et al., FEBS Lett. 408:285-288,1997. Analytical HPLC and mass spectrometry are used to assess thepurity of each peptide (>80%), which are readily soluble in PBS. On theday of injection, the peptide mixture is reconstituted in an 0.3 mg/mlalbumin solution (containing 4 mg/ml of phenol) (ALK/Abello, Horsholm,Denmark) and injected subcutaneously in the deltoid area.

Reagents: Whole birch pollen and Bet v 1 is purchased. For cell culture,Bet v 1 is further purified by HPLC. Its cytotoxicity can be inhibitedby overnight reduction at 37° C. with a 100 molar excess ofdithiothreitol, followed by alkylation with a 1000 molar excess ofN-ethylmaleimide. Bet v 1 is finally purified on a Sephadex G-25 column(Pharmacia, Uppsala, Sweden). PMA and ionomycin are purchased fromCalbiochem, San Diego, Calif.

Proliferation assays: Blood is drawn immediately before each OPFinjection and PBMC are isolated from heparinized blood by densitygradient centrifugation over Ficoll-Paque (Pharmacia Biotech AB,Uppsala, Sweden). Prior to ³H-thymidine (Du Pont NEN Products Boston,Mass., USA) incorporation, PBMC (2×10⁵/well) from each donor is culturedfor 6 days in octoplicates in 96 well flat bottom plates (Costar CorningInc., New York, N.Y.) in RPMI 1640 medium (Gibco, Basel, Switzerland)containing 10% AB⁺ serum (Swiss Red Cross, Bern, Switzerland), 2 mMglutamine, 1% Na-pyruvate, 1% non-essential amino acids, 1% kanamycine(all from Gibco) with optimal concentration of OPFs (10 μg/ml) or Bet v1 (10 μg/ml). See Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997.

Short term T cell lines: T cell lines are derived from PBMC that isisolated before each injection and stimulated in 24 well plates (Nunc)(10⁶ cells/well) with a mixture of the two OPFs (10 μg/ml) for 7 days insupplemented 10% AB⁺ RPMI 1640 medium as described above. The short termT cell lines obtained are washed and restimulated for 24 h (for IL-4,IL-5, IL-13 and TGFβ secretion) or 48 h (for IFNγ and IL-10) withplastic crosslinked OKT3 (1 μg/ml) (see Jutel et al., Clin. Experiment.Allergy 25:1108-1117, 1995). Cell culture supernatants are collected forcytokine quantification and stored at −80° C.

Cytokine quantification: IL-4, IL-10 and IFNγ are titrated usingcommercially available ELISA kits (Mabtech AG, Nacka, Sweden, for IL-4,IL-10 and IFNγ □□ and R&DSystem for IL-5, IL-13 and TGFβ), according tomanufacturer's recommendations.

Quantification of specific serum IgE and IgG₄: Whole birch pollen andanti-Bet v 1 specific IgE will be quantified using the Phamarcia CAPSystem Fluoroimmunoassay (Pharmacia Diagnostic AB, Uppsala, Sweden) asdescribed in Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997. For quantification of specific anti-Bet v 1 IgG4, native Bet v 1(5 μg/ml) is coated on 96 well plates (Maxisorb, Denmark) incarbonate/bicarbonate buffer pH 9.6, for 2 h at room temperature. Platesare blocked with milk 5%/PBS/Tween 0.05%. Serial dilutions of sera in 1%milk/Tween 0.05% are incubated for 1 h at room temperature. Plates arewashed thrice, incubated with horseradish peroxidase labelled anti-IgG4mAb JDC-14 1/10,000 (Pharmingen, Hamburg, Germany), and revealed in3,3′, 5,5′-tétraméthylbenzidine (TMB). Optical density is determined at450 nm on a microtiter plate analyzer (MR5000, Dynatech Laboratories).Titers are reported to a standard serum and expressed as arbitrarystandard units.

Immunoblotting and dot blot analysis: Anti-birch pollen or -Bet v1immunoblots will be processed as described in Kettner et al., Clin.Experiment. Allergy 29:394-401, 1999. For dot blot analysis, 1 μg ofwhole birch pollen, Bet v 1, OPFs or human albumin will be diluted ¼ inDMSO, spotted on PVDF membranes and dried for 30 min. at 37° C. Afterblocking in non-fat milk 5%, further steps are performed as described inKettner et al., Clin. Experiment. Allergy 29:394-401, 1999. Dotdensities are analyzed by scanning densitometry using an AdvancedAmerican Biotechnology scanner, Fullerton, Calif.

Statistical analysis: Differences within and between groups areevaluated by non-parametric ANOVA tests (Friedman or Kruskal-Wallis nonparametric test with multicomparison post-test, or Mann-Whitney testrespectively); or by Fisher's exact test (between group differences:responders versus non-responders, positive responses being defined as adoubling of day 0 value), using an Instat 3.0 software.

Example 3 Birch Pollen Profilin (Bet v 2) Specific T Cell ToleranceInduction with Allergen-Derived Overlapping Peptide Fragments

Materials and Methods

Patients: Patients eligible for this study include those with a historyof seasonal birch pollen allergy and with an SPT reaction ≧3+ comparedwith an albumin 10 mg/mL wheal and a minimal outcome of more than 3 mmwheal to commercial birch pollen extract.

Skin testing: Concentrations tested range from 10⁻³ μg/ml to 1 μg/ml(10-fold dilution series). An ID test result will be considered positivewhen a wheal reaction superior to 5 mm (for birch pollen profilin, Bet v2 and peptides) in diameter and an erythema were present at aconcentration ≦0.1 μg/ml.

Study design: The study is designed as a double blind, randomized,two-dose, placebo-controlled trial. At day 0, patients from the OPFgroup are injected at 30 min interval with successively 0.1 μg, 1 μg, 10μg, 20 μg, 40 μg, 80 μg and 100 μg of each of the two OPFs. Patients arethen injected at day 4, 7, 14, 42 and 70 with a maintenance dose of 100μg of each of the two OPFs. A maintenance dose of 300 μg of each OPF isinitially injected to two patients up to day 42. Patients from thecontrol group are injected with an equivalent volume of peptide diluentonly (0.3 mg/ml albumin solution, containing 4 mg/ml of phenol)(ALK/Abello, Horsholm, Denmark).

Peptide synthesis and purification: Two overlapping peptide fragmentsOPF₁₋₇₀ (SEQ ID NO:8) and OPF₆₀₋₁₃₃ (SEQ ID NO:9) mapping the entire 133amino acids of Bet v 2 (SEQ ID NO: 10) are synthesized on an AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.)and purified as described in Roggero et al., FEBS Lett. 408:285-288,1997. Analytical HPLC and mass spectrometry are used to assess thepurity of each peptide (>80%), which are readily soluble in PBS. On theday of injection, the peptide mixture is reconstituted in an 0.3 mg/mlalbumin solution (containing 4 mg/ml of phenol) (ALK/Abello, Horsholm,Denmark) and injected subcutaneously in the deltoid area.

Reagents: Whole birch pollen profilin and Bet v 2 is purchased. For cellculture, Bet v 2 is further purified by HPLC. Its cytotoxicity can beinhibited by overnight reduction at 37° C. with a 100 molar excess ofdithiothreitol, followed by alkylation with a 1000 molar excess ofN-ethylmaleimide. Bet v 1 is finally purified on a Sephadex G-25 column(Pharmacia, Uppsala, Sweden). PMA and ionomycin are purchased fromCalbiochem, San Diego, Calif.

Proliferation assays: Blood is drawn immediately before each OPFinjection and PBMC are isolated from heparinized blood by densitygradient centrifugation over Ficoll-Paque (Pharmacia Biotech AB,Uppsala, Sweden). Prior to ³H-thymidine (Du Pont NEN Products Boston,Mass., USA) incorporation, PBMC (2×10⁵/well) from each donor is culturedfor 6 days in octoplicates in 96 well flat bottom plates (Costar CorningInc., New York, N.Y.) in RPMI 1640 medium (Gibco, Basel, Switzerland)containing 10% AB⁺ serum (Swiss Red Cross, Bern, Switzerland), 2 mMglutamine, 1% Na-pyruvate, 1% non-essential amino acids, 1% kanamycine(all from Gibco) with optimal concentration of OPFs (10 μg/ml) or Bet v1 (10 μg/ml). See Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997.

Short term T cell lines: T cell lines are derived from PBMC that isisolated before each injection and stimulated in 24 well plates (Nunc)(10⁶ cells/well) with a mixture of the two OPFs (10 μg/ml) for 7 days insupplemented 10% AB⁺ RPMI 1640 medium as described above. The short termT cell lines obtained are washed and restimulated for 24 h (for IL-4,IL-5, IL-13 and TGFβ secretion) or 48 h (for IFNγ and IL-10) withplastic crosslinked OKT3 (1 μg/ml) (see Jutel et al., Clin. Experiment.Allergy 25:1108-1117, 1995). Cell culture supernatants are collected forcytokine quantification and stored at −80° C.

Cytokine quantification: IL-4, IL-10 and IFNγ are titrated usingcommercially available ELISA kits (Mabtech AG, Nacka, Sweden, for IL-4,IL-10 and IFNγ □□ and R&DSystem for IL-5, IL-13 and TGFβ), according tomanufacturer's recommendations.

Quantification of specific serum IgE and IgG₄: Whole birch pollenprofilin and anti-Bet v 2 specific IgE will be quantified using thePhamarcia CAP System Fluoroimmunoassay (Pharmacia Diagnostic AB,Uppsala, Sweden) as described in Kämmerer et al., J. Allergy Clin.Immunol. 100:96-103, 1997. For quantification of specific anti-Bet v 1IgG4, native Bet v 2 (5 μg/ml) is coated on 96 well plates (Maxisorb,Denmark) in carbonate/bicarbonate buffer pH 9.6, for 2 h at roomtemperature. Plates are blocked with milk 5%/PBS/Tween 0.05%. Serialdilutions of sera in 1% milk/Tween 0.05% are incubated for 1 h at roomtemperature. Plates are washed thrice, incubated with horseradishperoxidase labelled anti-IgG4 mAb JDC-14 1/10,000 (Pharmingen, Hamburg,Germany), and revealed in 3,3′, 5,5′-tétraméthylbenzidine (TMB). Opticaldensity is determined at 450 nm on a microtiter plate analyzer (MR5000,Dynatech Laboratories). Titers are reported to a standard serum andexpressed as arbitrary standard units.

Immunoblotting and dot blot analysis: Anti-birch pollen profilin or -Betv 2 immunoblots will be processed as described in Kettner et al., Clin.Experiment. Allergy 29:394-401, 1999. For dot blot analysis, 1 μg ofwhole birch pollen profilin, Bet v 2, OPFs or human albumin will bediluted ¼ in DMSO, spotted on PVDF membranes and dried for 30 min. at37° C. After blocking in non-fat milk 5%, further steps are performed asdescribed in Kettner et al., Clin. Experiment. Allergy 29:394-401, 1999.Dot densities are analyzed by scanning densitometry using an AdvancedAmerican Biotechnology scanner, Fullerton, Calif.

Statistical analysis: Differences within and between groups areevaluated by non-parametric ANOVA tests (Friedman or Kruskal-Wallis nonparametric test with multicomparison post-test, or Mann-Whitney testrespectively); or by Fisher's exact test (between group differences:responders versus non-responders, positive responses being defined as adoubling of day 0 value), using an Instat 3.0 software.

Example 4 Dust Mite (Der p 1) Specific T Cell Tolerance Induction withAllergen-Derived Overlapping Peptide Fragments

Materials and Methods

Patients: Patients eligible for this study include those with a historyof dust mite allergy and with an SPT reaction ≧3+ compared with analbumin 10 mg/mL wheal and a minimal outcome of more than 3 mm wheal tocommercial dust mite extract.

Skin testing: Concentrations tested range from 10⁻³ μg/ml to 1 μg/ml(10-fold dilution series). An ID test result will be considered positivewhen a wheal reaction superior to 5 mm (for dust mite, Der p 1 andpeptides) in diameter and an erythema were present at a concentration≦0.1 μg/ml.

Study design: The study is designed as a double blind, randomized,two-dose, placebo-controlled trial. At day 0, patients from the OPFgroup are injected at 30 min interval with successively 0.1 μg, 1 μg, 10μg, 20 μg, 40 μg, 80 μg and 100 μg of each of the two OPFs. Patients arethen injected at day 4, 7, 14, 42 and 70 with a maintenance dose of 100μg of each of the three OPFs. A maintenance dose of 300 μg of each OPFis initially injected to two patients up to day 42. Patients from thecontrol group are injected with an equivalent volume of peptide diluentonly (0.3 mg/ml albumin solution, containing 4 mg/ml of phenol)(ALK/Abello, Horsholm, Denmark).

Peptide synthesis and purification: Three overlapping peptide fragmentsOPF₁₋₈₁ (SEQ ID NO:11), OPF₆₇₋₁₅₂ (SEQ ID NO:12) and OPF₁₃₇₋₂₁₂ (SEQ IDNO:13) mapping the entire 212 amino acids of Der p 1 (SEQ ID NO: 14) aresynthesized on an Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) and purified as described in Roggero et al.,FEBS Lett. 408:285-288, 1997. Analytical HPLC and mass spectrometry areused to assess the purity of each peptide (>80%), which are readilysoluble in PBS. On the day of injection, the peptide mixture isreconstituted in an 0.3 mg/ml albumin solution (containing 4 mg/ml ofphenol) (ALK/Abello, Horsholm, Denmark) and injected subcutaneously inthe deltoid area.

Reagents: Whole DM and Der p 1 is purchased. For cell culture, Der p 1is further purified by HPLC. Its cytotoxicity can be inhibited byovernight reduction at 37° C. with a 100 molar excess of dithiothreitol,followed by alkylation with a 1000 molar excess of N-ethylmaleimide. Derp 1 is finally purified on a Sephadex G-25 column (Pharmacia, Uppsala,Sweden). PMA and ionomycin are purchased from Calbiochem, San Diego,Calif.

Proliferation assays: Blood is drawn immediately before each OPFinjection and PBMC are isolated from heparinized blood by densitygradient centrifugation over Ficoll-Paque (Pharmacia Biotech AB,Uppsala, Sweden). Prior to ³H-thymidine (Du Pont NEN Products Boston,Mass., USA) incorporation, PBMC (2×10⁵/well) from each donor is culturedfor 6 days in octoplicates in 96 well flat bottom plates (Costar CorningInc., New York, N.Y.) in RPMI 1640 medium (Gibco, Basel, Switzerland)containing 10% AB⁺ serum (Swiss Red Cross, Bern, Switzerland), 2 mMglutamine, 1% Na-pyruvate, 1% non-essential amino acids, 1% kanamycine(all from Gibco) with optimal concentration of OPFs (10 μg/ml) or Bet v1 (10 μg/ml). See Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997.

Short term T cell lines: T cell lines are derived from PBMC that isisolated before each injection and stimulated in 24 well plates (Nunc)(10⁶ cells/well) with a mixture of the three OPFs (10 μg/ml) for 7 daysin supplemented 10% AB⁺ RPMI 1640 medium as described above. The shortterm T cell lines obtained are washed and restimulated for 24 h (forIL-4, IL-5, IL-13 and TGFβ secretion) or 48 h (for IFNγ and IL-10) withplastic crosslinked OKT3 (1 μg/ml) (see Jutel et al., Clin. Experiment.Allergy 25:1108-1117, 1995). Cell culture supernatants are collected forcytokine quantification and stored at −80° C.

Cytokine quantification: IL-4, IL-10 and IFNγ are titrated usingcommercially available ELISA kits (Mabtech AG, Nacka, Sweden, for IL-4,IL-10 and IFNγ □□ and R&DSystem for IL-5, IL-13 and TGFβ), according tomanufacturer's recommendations.

Quantification of specific serum IgE and IgG₄: Whole DM and anti-Der p 1specific IgE will be quantified using the Phamarcia CAP SystemFluoroimmunoassay (Pharmacia Diagnostic AB, Uppsala, Sweden) asdescribed in Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997. For quantification of specific anti-Der p 1 IgG4, native Der p 1(5 μg/ml) is coated on 96 well plates (Maxisorb, Denmark) incarbonate/bicarbonate buffer pH 9.6, for 2 h at room temperature. Platesare blocked with milk 5%/PBS/Tween 0.05%. Serial dilutions of sera in 1%milk/Tween 0.05% are incubated for 1 h at room temperature. Plates arewashed thrice, incubated with horseradish peroxidase labelled anti-IgG4mAb JDC-14 1/10,000 (Pharmingen, Hamburg, Germany), and revealed in3,3′, 5,5′-tétraméthylbenzidine (TMB). Optical density is determined at450 nm on a microtiter plate analyzer (MR5000, Dynatech Laboratories).Titers are reported to a standard serum and expressed as arbitrarystandard units.

Immunoblotting and dot blot analysis: Anti-DM or -Der p 1 immunoblotswill be processed as described in Kettner et al., Clin. Experiment.Allergy 29:394-401, 1999. For dot blot analysis, 1 μg of dust miteallergen, Der p 1, OPFs or human albumin will be diluted ¼ in DMSO,spotted on PVDF membranes and dried for 30 min. at 37° C. After blockingin non-fat milk 5%, further steps are performed as described in Kettneret al., Clin. Experiment. Allergy 29:394-401, 1999. Dot densities areanalyzed by scanning densitometry using an Advanced AmericanBiotechnology scanner, Fullerton, Calif.

Statistical analysis: Differences within and between groups areevaluated by non-parametric ANOVA tests (Friedman or Kruskal-Wallis nonparametric test with multicomparison post-test, or Mann-Whitney testrespectively); or by Fisher's exact test (between group differences:responders versus non-responders, positive responses being defined as adoubling of day 0 value), using an Instat 3.0 software.

Example 5 Dust Mite (Der p 2) Specific T Cell Tolerance Induction withAllergen-Derived Overlapping Peptide Fragments

Materials and Methods

Patients: Patients eligible for this study include those with a historyof dust mite allergy and with an SPT reaction ≧3+ compared with analbumin 10 mg/mL wheal and a minimal outcome of more than 3 mm wheal tocommercial dust mite extract.

Skin testing: Concentrations tested range from 10⁻³ μg/ml to 1 μg/ml(10-fold dilution series). An ID test result will be considered positivewhen a wheal reaction superior to 5 mm (for dust mite, Der p 2 andpeptides) in diameter and an erythema were present at a concentration≦0.1 μg/ml.

Study design: The study is designed as a double blind, randomized,two-dose, placebo-controlled trial. At day 0, patients from the OPFgroup are injected at 30 min interval with successively 0.1 μg, 1 μg, 10μg, 20 μg, 40 μg, 80 μg and 100 μg of each of the two OPFs. Patients arethen injected at day 4, 7, 14, 42 and 70 with a maintenance dose of 100μg of each of the three OPFs. A maintenance dose of 300 μg of each OPFis initially injected to two patients up to day 42. Patients from thecontrol group are injected with an equivalent volume of peptide diluentonly (0.3 mg/ml albumin solution, containing 4 mg/ml of phenol)(ALK/Abello, Horsholm, Denmark).

Peptide synthesis and purification: Two overlapping peptide fragmentsOPF₁₋₇₃ (SEQ ID NO:15) and OPF₅₇₋₁₃₆ (SEQ ID NO:16) mapping the entire136 amino acids of Der p 2 (SEQ ID NO: 17) are synthesized on an AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.)and purified as described in Roggero et al., FEBS Lett. 408:285-288,1997. Analytical HPLC and mass spectrometry are used to assess thepurity of each peptide (>80%), which are readily soluble in PBS. On theday of injection, the peptide mixture is reconstituted in an 0.3 mg/mlalbumin solution (containing 4 mg/ml of phenol) (ALK/Abello, Horsholm,Denmark) and injected subcutaneously in the deltoid area.

Reagents: Whole DM and Der p 2 is purchased. For cell culture, Der p 2is further purified by HPLC. Its cytotoxicity can be inhibited byovernight reduction at 37° C. with a 100 molar excess of dithiothreitol,followed by alkylation with a 1000 molar excess of N-ethylmaleimide. Derp 2 is finally purified on a Sephadex G-25 column (Pharmacia, Uppsala,Sweden). PMA and ionomycin are purchased from Calbiochem, San Diego,Calif.

Proliferation assays: Blood is drawn immediately before each OPFinjection and PBMC are isolated from heparinized blood by densitygradient centrifugation over Ficoll-Paque (Pharmacia Biotech AB,Uppsala, Sweden). Prior to ³H-thymidine (Du Pont NEN Products Boston,Mass., USA) incorporation, PBMC (2×10⁵/well) from each donor is culturedfor 6 days in octoplicates in 96 well flat bottom plates (Costar CorningInc., New York, N.Y.) in RPMI 1640 medium (Gibco, Basel, Switzerland)containing 10% AB⁺ serum (Swiss Red Cross, Bern, Switzerland), 2 mMglutamine, 1% Na-pyruvate, 1% non-essential amino acids, 1% kanamycine(all from Gibco) with optimal concentration of OPFs (10 μg/ml) or Bet v1 (10 μg/ml). See Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997.

Short term T cell lines: T cell lines are derived from PBMC that isisolated before each injection and stimulated in 24 well plates (Nunc)(10⁶ cells/well) with a mixture of the two OPFs (10 μg/ml) for 7 days insupplemented 10% AB⁺ RPMI 1640 medium as described above. The short termT cell lines obtained are washed and restimulated for 24 h (for IL-4,IL-5, IL-13 and TGFβ secretion) or 48 h (for IFNγ and IL-10) withplastic crosslinked OKT3 (1 μg/ml) (see Jutel et al., Clin. Experiment.Allergy 25:1108-1117, 1995). Cell culture supernatants are collected forcytokine quantification and stored at −80° C.

Cytokine quantification: IL-4, IL-10 and IFNγ are titrated usingcommercially available ELISA kits (Mabtech AG, Nacka, Sweden, for IL-4,IL-10 and IFNγ □□ and R&DSystem for IL-5, IL-13 and TGFβ), according tomanufacturer's recommendations.

Quantification of specific serum IgE and IgG₄: Whole DM and anti-Der p 2specific IgE will be quantified using the Phamarcia CAP SystemFluoroimmunoassay (Pharmacia Diagnostic AB, Uppsala, Sweden) asdescribed in Kämmerer et al., J. Allergy Clin. Immunol. 100:96-103,1997. For quantification of specific anti-Der p 2 IgG4, native Der p 2(5 μg/ml) is coated on 96 well plates (Maxisorb, Denmark) incarbonate/bicarbonate buffer pH 9.6, for 2 h at room temperature. Platesare blocked with milk 5%/PBS/Tween 0.05%. Serial dilutions of sera in 1%milk/Tween 0.05% are incubated for 1 h at room temperature. Plates arewashed thrice, incubated with horseradish peroxidase labelled anti-IgG4mAb JDC-14 1/10,000(Pharmingen, Hamburg, Germany), and revealed in 3,3′,5,5′-tétraméthylbenzidine (TMB). Optical density is determined at 450 nmon a microtiter plate analyzer (MR5000, Dynatech Laboratories). Titersare reported to a standard serum and expressed as arbitrary standardunits.

Immunoblotting and dot blot analysis: Anti-DM or -Der p 2 immunoblotswill be processed as described in Kettner et al., Clin. Experiment.Allergy 29:394-401, 1999. For dot blot analysis, 1 μg of dust miteallergen, Der p 2, OPFs or human albumin will be diluted ¼ in DMSO,spotted on PVDF membranes and dried for 30 min. at 37° C. After blockingin non-fat milk 5%, further steps are performed as described in Kettneret al., Clin. Experiment. Allergy 29:394-401, 1999. Dot densities areanalyzed by scanning densitometry using an Advanced AmericanBiotechnology scanner, Fullerton, Calif.

Statistical analysis: Differences within and between groups areevaluated by non-parametric ANOVA tests (Friedman or Kruskal-Wallis nonparametric test with multicomparison post-test, or Mann-Whitney testrespectively); or by Fisher's exact test (between group differences:responders versus non-responders, positive responses being defined as adoubling of day 0 value), using an Instat 3.0 software.

OTHER EMBODIMENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique methods andcompositions have been described. Although particular embodiments havebeen disclosed herein in detail, this has been done by way of examplefor purposes of illustration only, and is not intended to be limitingwith respect to the scope of the appended claims that follow. Inparticular, it is contemplated by the inventor that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

What is claimed is:
 1. A composition comprising a plurality ofcontiguous overlapping peptide fragments 30 to 73 amino acids in lengthwhich peptide fragments consist of peptides within SEQ ID NOs: 15 and 16which together comprise the entire amino acid sequence of a dust miteallergen (SEQ ID NO: 17), wherein said fragments are capable of inducinga T cell response in patients who are hypersensitive to said allergenwherein the reactivity of said peptides to IgE antibodies of subjectswho are allergic to dust mite allergen Der p 2 is significantly reducedor eliminated while the reactivity with the T lymphocytes from subjectswho are allergic to dust mite allergen Der p 2 is retained.